Adenosine triphosphate (hereinafter simply referred to as “ATP”) is the energy currency in living organisms.
Until now, the most commonly used measuring method of ATP has been luciferin-luciferase system and a small amount of ATP in a solution can be measured selectively with this system (Nonpatent Document Nos. 1 to 3). Reagents and kits regarding ATP measurement by luciferin-luciferase system already have been commercially available and commonly used. For example, LL100-1/ATP Luminescence Kit (TOYO INK GROUP), ATPlite ATP detection system (Perkin Elmer) and the like is included. However, in the case where luciferase is expressed in the cells to measure ATP concentration in a living organism, quantitative measurement is difficult and thus following the change in ATP concentration with a high temporal resolution is difficult, because of the drawbacks that luminescence of luciferase is dark and also the amount of luminescence is dependent on the concentration of protein.
Further, since luciferase has an ATP-hydrolytic activity, it can be considered that expressing luciferase in cells may change ATP concentration.
Another method is high performance liquid chromatography (HPLC) method, where ATP is detected by separating it from other substances through the use of an appropriate column (Nonpatent Document No. 4). The method enables quantitative measurement of ATP in solution, but the operation is so complicated that measuring ATP in vivo, namely in living cells, is impossible.
Alternatively, measuring ATP by 31P-NMR method has also been reported (Nonpatent Document Nos. 5 to 7). In this approach, its merit is that ATP concentration in a living organism can be accurately obtained without forcedly expressing proteins such as luciferase. However, its poor spatial resolution and poor sensitivity limit the measurement only to the tissue level and its poor temporal resolution provides a drawback of requiring long measuring time. Further, extremely expensive measuring equipment is also a major drawback.
In reality, in order to measure ATP in a living organism, there are overwhelmingly many cases where the living organism is broken to obtain extract and the ATP in the extract is measured by luciferase or HPLC. However, in such a case where a living organism is broken to obtain extract and ATP in the extract is measured, there is a drawback that ATP is hydrolyzed before measurement.
As a new measuring method, a measuring method using fluorescence resonance energy transfer (fluorescence resonance energy transfer: FRET) technique has been reported where cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) are attached to CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2), which is a protein reactive with adenosine nucleic acids such as ATP, ADP and AMP (Nonpatent Document No. 8).
[Nonpatent Document No. 1] Appl Microbiol. 1975; 30, 713-721.
[Nonpatent Document No. 2] J. Am. Soc. Brew. Chem. 1976; 34, 145-150.
[Nonpatent Document No. 3] Plant Cell Physiol. 1979; 20, 145-155.
[Nonpatent Document No. 4] J. Mol. Cell. Cardiol. 1986; 18, 517-527.
[Nonpatent Document No. 5] Nature 1977; 265, 756-758.
[Nonpatent Document No. 6] Proc. Nat. Acad. Sci. U.S.A. 1979; 76, 7445-7449.
[Nonpatent Document No. 7] J. Biol. Chem. 1980; 225, 3987-3993
[Nonpatent Document No. 8] Nat. Biotechnol. 2007; 25 (2):170-172.